| 1. ObjectiveSince it was proved that the Tricula.sp can be the snail hosts of Schistosomiasis mekongi, Schistosomiasis sinensium and Paragonimiasis skrjabini, the significance of Tricula has been recognized. Recently, some reports said the Paragonimiasis has a resurgent trend in southeast China and some inland cities. As the first intermediate hosts of Pagumogonimus skrjabini, the classification of Tricula not only has great significance in biology, but also beneficial to the medicine practice. Until now, more than 100 kinds of species belong to Triculinae were reported in Asia and half of them located in China. The Tricula species were identified by the traditional means. But there are many disputes existed in such a classification. With the development of molecule biological, some new techniques were provided to us. The RAPD technique was used in this study to analyze the genetic variation of 6 populations of Tricula collected from 5 areas of 3 provinces. The exterior morphological features and ecological environment are fundamental factors to identify a new species, so the exterior morphological features were observed and the ecological environment were analyzed in this research also.2. Materials and methods6 tricula populations were collected from 5 areas of 3 provinces in China, such as Guangxi-Wuming, Fujian-Fuzhou, Hubei-Wufeng, Hubei-Shiyan and Hubei-Zhongxiang. Tricula species collected from Fujian-Fuzhou, Hubei-Wufeng and Hubei-Zhongxiang were reported formally and named T.xindiangensis, T.pingi, T.hsiangi and T.hongshanensis respectively. The populations collected from Guangxi-Wuming and Hubei-Shiyan was called Guangxi-Wuming isolate and Hubei-Shiyan isolate in this research. 1. Make a systematic observation of the exterior morphological features by naked eyes and stereo microscope, comparing the ecological environment of the snails and record the results. 2. High-Content-NaCl-Solution method was used to extract the genomic DNA. RAPD technique was used to amplified the genomic DNA. Then, the genetic distance & genetic similarity of the tricula from various regions were calculated according to the types of bands of the amplified products on the agarose gel electrophoresis. The phylogenetic tree was thereby mapped, and the DNA polymorphic bands detected were analyzed by clustering analysis with spss11.0 software.3. Results①Observational and comparative of exterior morphological features and ecological environments of different population of Tricula:There are much similarity between T. pingi and T. hsiangi, T. hongshanensis and T. xindiangensis in the exterior morphological features and the habitat environments. While other species show much diversity.②DNA extraction:The A260/A280 value of the DNA solution ranged from 1.60～2.00, the purity is qualified.③RAPD:1) The difference out of species: The RAPD products of 6 Tricula populations and Semisulcospira cancellata showed obvious dissimilarity. 87 bands, which were homologous to 87 loci of the genome, were amplified with 6 random primers. There are 5 bands of the 87 bands were the same between the two species. The genetic distance between them was larger than 0.66, and the genetic similarity was only 0.339.2) The difference between species: Genetic distances among the 6 Tricula populations ranged from 0.1301～0.3151, and the average was 0.2248. The type of bands of amplified products between T.pingi and T.hsiangi were much similar. The genetic similarity was 0.8699 and the genetic distance was only 0.1301.The bands type of T. hongshanensis and T. xindiangensis were much similar. The genetic similarity was 0.8558 and the genetic distance was only 0.1442. The bands type of these two snails had some similarity with Hubei-Shiyan isolate in some degree. While Guangxi-Wuming isolate had much diversity to others. The genetic distance among them ranged from 0.2234～0.3151.3) The populations of Tricula involved in this article can be divided into four groups:①T. pingi and T. hsiangi;②T. hongshanensis and T. xindiangensis;③Hubei-Shiyan isolates;④G uangxi-Wuming isolates.4. ConclusionAccording to the results of RAPD-PCR, 6 populations of Tricula involved in this article can be divided into four groups in terms of genetic level:①T. pingi and T. hsiangi;②T. hongshanensis and T. xindiangensis;③Hubei-Shiyan isolates;④G uangxi-Wuming isolates. Differences at genetic level exist in Tricula from different areas in China, such genetic differences in accord with the exterior morphological features and the ecological environment of the Tricula to some degree.This research firstly used the RAPD technique to analyze the genetic variation of Tricula. In addition, exterior morphological features were observed and the ecological environments of different population of Tricula were analyzed. The classification by the RAPD in this research was in accord with the traditional classification in principle. The RAPD technique was an effective method to analyze the genetic variation of Tricula.
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